Four independent hematopoietic precursor cell types are specified during development, culminating in specification of hematopoietic stem cells (HSCs), which self-renew and provide all of the major blood lineages over the lifetime of an adult organism. The first three precursor cell types are transient progenitors believed to temporarily provide blood and immune cells to the embryo until HSCs finally emerge. Precisely where and when HSCs are specified has until recently been highly controversial, but recent studies have conclusively demonstrated that they arise from hemogenic endothelium, a special population of endothelial cells within the ventral wall of the primitive dorsal aorta that transdifferentiate into HSCs. In our previous studies, we imaged directly the birth of HSCs from ventral aortic endothelium for the first time. Complementary lineage tracing of these HSC founders indicated that they provide all adult hematopoietic cells, and are thus the unique source of all HSCs. The signaling events underlying the developmental specification of hemogenic endothelium remain poorly understood. In our recent studies, we utilized the unique experimental advantages afforded by the zebrafish embryo to uncover a novel model of HSC induction, where Wnt16 function in the ventral somite is required for HSC specification. The HSC defect in Wnt16 morphants could be rescued by enforced Notch signaling, but only within an early window of development, well before HSC precursors experience Notch signaling. This environmental Notch requirement is mediated by the Notch ligands DeltaC and DeltaD, but how these connect to Notch signaling within the precursors of HSCs is unknown. Our current studies demonstrate that three of the four Notch receptors are required to establish HSC fate. The focus of our continuing efforts is to determine where and when each of these requirements are needed, to determine whether or not each is required in a cell-autonomous manner, to determine which Notch ligands are utilized to regulate each signaling event, and to determine functionally when HSC fate is established through lineage tracing and cell transplantation assays. With the elucidation of this signaling axis, and the cellular behaviors it controls, our wok will ultimately enable ex vivo approaches to direct patient-specific iPS cells towards the HSC fate for cellular replacement therapies.